Real-time and quantitative imaging of mammalian stress granules and processing bodies

Nuclear mRNA domains such as nucleoli, speckles, Cajal bodies, and gems demonstrate that RNA function and morphology are inextricably linked; granular mRNA structures are self-generated in tandem with metabolic activity. Similarly, cytoplasmic compartmentalization of mRNA into mRNP structures such as stress granules (SGs) and processing bodies (PBs) reiterate the link between function and structure; the assembly of SGs and PBs requires mRNA released from disassembling polysomes on translational arrest. SGs contain mRNA still associated with some of the translational machinery, specifically 40S subunits and a subset of translation initiation factors including eIF3, eIF4F, eIF4B, and PABP. PBs also contain mRNA and eIF4E but lack other preinitiation factors and contain instead a number of proteins associated with mRNA decay such as DCP1a, DCP2, hedls/GE-1, p54/RCK. Many other proteins (e.g., argonaute, FAST, RAP-55, TTP) and microRNAs are present in both SGs and PBs, sometimes shepherding specific mRNA transcripts between the translation and decay machineries. Recently, we described markers and methods to visualize SGs and PBs in fixed cells (Kedersha and Anderson, 2007), but understanding the dynamic nature of SGs and PBs requires live cell imaging. This presents unique challenges, because it requires the overexpression of fluorescently tagged SG/PB marker proteins, which can shift the mRNA equilibrium toward SGs or PBs, thus obscuring the result. We describe stably expressed, fluorescently tagged SG and PB markers that exhibit similar behavior to their endogenous counterparts, thus allowing real-time imaging of SGs and PBs.